JP2018515622A - Synergistic combination of neuronal survival factors and uses thereof - Google Patents

Abstract

The present invention relates to a synergistic combination of short and long rod-derived cone survival factors, and methods for treating retinal degenerative diseases.

Description

The present invention relates to neurodegenerative disorders, and more particularly to pharmaceutical compositions for treating and / or preventing neurodegenerative disorders.

BACKGROUND OF THE INVENTION Neurodegenerative disorders include a wide range of severe debilitating conditions characterized by neuronal degeneration.

  Rod cone dystrophy, such as retinitis pigmentosa (RP), is a genetically heterogeneous retinal degenerative disease characterized by progressive death of rod photoreceptors and subsequent loss of cones. is there. RP is one of the most common forms of hereditary retinal degeneration, affecting approximately 1: 3,500 people worldwide (1). Over 54 mutations that cause RP have been identified so far, most of these mutations being in rod-specific transcripts. RP patients initially exhibit a loss of vision in low light environments as a result of rod dysfunction, and the associated conservation of vision, mediated by the macular cone. However, as the disease progresses, primary loss of rods is followed by cone degeneration and a corresponding lack of cone-mediated vision. In modern societies where most of the environment is artificial lighting and many activities rely on high-intelligence color vision, maintaining cone-mediated vision in RP patients will lead to a significant improvement in quality of life .

  The loss of cones in the RP subset caused by rod-specific mutations has been largely unclear, but several mechanisms have been proposed that are not necessarily mutually exclusive. Some hypothesized mechanisms are that cone death may result from degeneration of the retinal pigment epithelium (RPE) or Mueller glia as a result of loss of contact with or in the surrounding rod. The “adjacent effect” is implied as a result of endotoxin release. Alternatively, Müller cell activation and release of toxic molecules may play a role. Another hypothesis is that as the metabolic burden of the rod is lost, the amount of oxygen or retinoid delivered by the RPE from the choroidal blood circulation to the photoreceptor layer becomes excessive and toxic (2). Punzo et al. Showed evidence that cones partially die as a result of starvation and nutritional imbalance driven by an insulin / mammalian target of the rapamycin pathway in a murine model of retinal degeneration (3). In addition, it has been suggested that loss of survival factors secreted by the rod and necessary for cone survival can contribute to cone loss (4, 5).

  Consistent with the final hypothesis, in rd1 mice, transplanted healthy retinal tissue has been shown to support cone survival in areas distant from the grafted tissue (6, 7).

  International Patent Application WO2008 / 148860A1 is a rod derived cone survival factor (RdCVF) that can increase neuronal survival and is useful for treating and / or preventing neurodegenerative disorders such as RP and It describes a family of trophic factors called RdCVF2.

  Rod-derived cone survival factor (RdCVF) was initially identified as a candidate molecule responsible for this rescue effect from a high-throughput method of screening a cDNA library (4). Rods secrete RdCVF, so as the rods die, this paracrine factor source is lost and RdCVF levels decrease. Loss of expression of RdCVF and similar secreted factors may therefore contribute to the second wave of cone degeneration observed in rod cone dystrophy. RdCVF has been shown to mediate cone survival both in culture (8) and subretinal injections in mice and rat models of recessive and dominant forms of retinitis pigmentosa (4,9). Disruption of NXNL1 (the gene encoding RdCVF) makes mouse photoreceptors increasingly sensitive to photoreceptor dysfunction and cone loss over time (10).

  NXNL1 encodes two isoforms of RdCVF through alternative splicing. The isoform that mediates cone survival is the longer truncated form of the pair (RdCVFL) and contains a C-terminal extension that confers enzyme function (11). RdCVFL, containing all of the amino acids of RdCVF, is encoded by exons 1 and 2 of the NXNL1 gene and is a member of the thioredoxin family (12). Thioredoxins have a variety of functions including maintaining an appropriate reducing environment in the cell and participating in the apoptotic pathway. These functions are achieved through a thioloxidoreductase reaction mediated by a conserved CXXXC catalytic site within the thioredoxin fold (13).

  However, there is still a need for additional neuroprotective treatment for neurodegenerative disorders.

Summary of the Invention The present inventors have surprisingly found that a synergistic effect could be obtained by combining a short isoform (RdCVF) and a long isoform (RdCVFL) of the NXNL1 gene.

  Here, the present invention is a method for treating a patient suffering from a retinal degenerative disease, wherein the patient is treated with a therapeutically effective amount of a short isoform of the NXNL1 gene, rod-derived cone survival factor (RdCVF). And a second nucleic acid encoding a long isoform of the NXNL1 gene (RdCVFL).

  The short and long isoforms may be administered by separate vectors or by a single vector.

  Thus, in one aspect, the invention also relates to an expression vector comprising a first nucleic acid encoding a rod-derived cone survival factor (RdCVF) short isoform and a second nucleic acid encoding an RdCVF long isoform. .

In another embodiment, the present invention is also a kit of parts for use in a method for treating a photoreceptor degenerative disorder comprising:
A first expression vector comprising a first nucleic acid encoding a short isoform [rod-derived cone survival factor (RdCVF)] and a second comprising a second nucleic acid encoding a long isoform (RdCVFL) The present invention relates to a kit of parts including the expression vector.

DETAILED DESCRIPTION OF THE INVENTION The present invention is a method for treating a patient suffering from a retinal degenerative disease, wherein the patient is treated with a therapeutically effective amount of a short isoform [rod-derived cone survival factor (RdCVF)]. And a second nucleic acid encoding a long isoform (RdCVF).

  The short and long isoforms may be administered by separate vectors or by a single vector.

  Accordingly, in a first aspect, the present invention provides a method for treating a patient suffering from retinal degenerative disease with a first nucleic acid encoding a therapeutically effective amount of a short isoform [rod-derived cone survival factor (RdCVF)] and a long nucleic acid. A method for treating said patient comprising the step of administering a second nucleic acid encoding an isoform (RdCVFL), wherein said first nucleic acid and said second nucleic acid are single expression It relates to a method contained in a vector.

  The present invention also relates to an expression vector comprising a first nucleic acid encoding a short isoform [rod-derived cone survival factor (RdCVF)] and a second nucleic acid encoding a long isoform (RdCVFL).

  As used herein, the term rod-derived cone survival factor (RdCVF) refers to a protein encoded by a thioredoxin-like 6 (Txnl6) or nucleredoxin-like 1 (NXNL1) gene. It includes RdCVF protein of any animal species. Typically, an RdCVF protein according to the invention can be a mammalian RdCVF protein, including but not limited to mice, rats, cats, dogs, non-human primates and humans.

  Unless otherwise specified, the term “RdCVF” refers to the short isoform of the NXNL1 gene, and the term “RdCVFL” or “RdCVF-L” refers to the long isoform of the NXNL1 gene.

  Typically, in mice, the short isoform (RdCVF) is a 109 amino acid long protein referenced under Uniprot accession number Q91W38. The murine long isoform (RdCVF-L) is a 217 amino acid long protein referenced under Q8VC33.

  In one embodiment of the invention, the short isoform of RdCVF is the short isoform of human RdCVF (hRdCVF) having the following sequence:

  In one embodiment of the invention, the long isoform of the NXNL1 gene is the long isoform of human RdCVFL (hRdCVFL), referenced under accession number Q96CM4 and having the sequence shown below:

  The sequences of RdCVF and RdCVFL proteins are described in Chalmel et al. 2007, BMC Molecular Biology 2007, 8:74 and International Patent Application WO2008 / 148860.

  As used herein, the terms “vector” and “expression vector” are used interchangeably and refer to an expression vector. The expression vector according to the present invention may be in the form of a plasmid, virus, phage or the like.

  Typically, the expression vector according to the present invention can be a virus.

  In one embodiment, the expression vector is an adeno-associated vector (AAV).

  AAV has been widely described in the art as a suitable vector for gene delivery. Indeed, AAV is non-pathogenic and presents a wide range of tissue specificities. Typically, the AAV according to the present invention is an AAV that can target retinal cells.

  Examples include but are not limited to AAV2, AAV2 / 8, AAV9 and AAV7m8.

  In one embodiment, the AAV according to the invention is obtained according to the method described in the international patent application WO2012 / 158757.

  In the context of the present invention, the terms “treating” or “treatment” as used herein refer to disorders or conditions to which such terms apply or such It means to reverse, alleviate or inhibit the progression of one or more symptoms of a disorder or condition (eg, retinal degenerative disease) or to prevent the disorder or condition or one or more symptoms thereof.

  The term “retinal degenerative disease” encompasses all diseases associated with cone degeneration. Retinal degenerative diseases include, but are not limited to, retinitis pigmentosa, age-related macular degeneration, Bardet-Biedel syndrome, Bassen-Kornzweig syndrome, Vest disease, choroidal atrophy, cerebral atrophy, Including Labor congenital cataract, Refsum disease, Stargardt disease or Usher syndrome.

  In one embodiment of the invention, the retinal degenerative disease is retinal pigment degeneration.

  According to the present invention, the term “patient” or “patient in need thereof” intends a human or non-human mammal suffering from or likely to have a retinal degenerative disease.

  According to the present invention, a “therapeutically effective amount” of a composition is a therapeutically effective amount that is sufficient to achieve the desired biological effect, in this case, to increase neuronal survival. It will be appreciated that the effective dosage will depend on the age, sex, health and weight of the recipient, the type of combination treatment, if any, the frequency of treatment, and the nature of the desired effect. However, preferred dosages can be tailored to individual subjects without undue experimentation, as will be understood and determinable by one of ordinary skill in the art.

  The expression vector of the present invention can be suitable for intravenous administration or intraocular administration. In certain embodiments, the expression vector is administered by intravitreal injection.

  In one aspect, the present invention also provides a first nucleic acid encoding a short isoform of the NXNL1 gene [rod-derived cone survival factor (RdCVF)] and a long isoform of the NXNL1 gene (RdCVF-L). The present invention relates to a pharmaceutical composition comprising an expression vector containing two nucleic acids and a pharmaceutically acceptable carrier.

  Without being bound by theory, it is believed that the delivery of short isoform (RdCVF) and long isoform (RdCVF-L) leads to a synergistic effect.

  On the other hand, the short isoform (RdCVF) is produced and secreted by the retinal pigment epithelium (RPE) and stimulates aerobic glycolysis through the RdCVF receptor on the cone cell surface by a non-autonomous mechanism Protect the cones.

  On the other hand, the long isoform (RdCVFL) protects the cone from cell-autonomous oxidative damage due to its thiooxide reductase function.

  In another aspect of the invention, the short and long isoforms (RdCVF and RdCVF-L) are administered by separate vectors, which can be administered simultaneously or sequentially.

  Therefore, the present invention also provides a patient suffering from retinal degenerative disease with a second nucleic acid encoding a therapeutically effective amount of a first nucleic acid encoding a short isoform (RdCVF) and a long isoform (RdCVF-L). A method for treating said patient comprising administering a nucleic acid, wherein said first nucleic acid and second nucleic acid are contained in separate expression vectors.

The invention also relates to a kit of parts for use in a method for treating photoreceptor degeneration disorders, comprising:
A first expression vector comprising a first nucleic acid encoding a short isoform of the NXNL1 gene [rod-derived cone survival factor (RdCVF)], and a long isoform of the NXNL1 gene (RdCVF-L) A second expression vector comprising a second nucleic acid.

  The invention will be further described through the following examples and figures.

Schematic of 2xRdCVF vector (plasmids p857 and AAV CT-39) kanamycin: plasmid selection in bacteria 5 'ITR AAV inverted terminal repeat CMV / CBA promoter delta 390: mixed cytomegalovirus / chicken beta actin (ubiquitous and strong promoter) hRdCVF: human RdCVF cDNA BGH poly A: mRNA stabilization pTF3, bla txn terminator, rpn txn terminator: a set of transcription terminators and transcription insulators located outside the 3 "ITR in the plasmid (ie not in the rAAV genome) PTF3 is incorporated into bla txn CMV / CBA promoter delta 390: mixed cytomegalovirus / chicken beta actin ( Standing manner strong promoter) hRdCVF: Human RdCVF cDNA BGH poly A: Stabilization 3 'ITR AAV inverted terminal repeats of the mRNA. Schematic of RdCVF-RdCVFL vector (plasmids p853 and AAV CT-35) The vector contains the following elements: kanamycin: plasmid selection in bacteria 5'ITR AAV inverted terminal repeats CMV / CBA promoter delta 390: mixing site Megalovirus / chicken beta actin (ubiquitous and strong promoter) hRdCVF: human RdCVF cDNA BGH poly A: mRNA stabilization pTF3, bla txn terminator, rpn txn terminator: located outside the 3 ”ITR in the plasmid (ie A set of transcription terminators and transcription insulators (not in the rAAV genome) pTF3 is integrated into bla txn CMV / CBA promoter delta 39 : Mixed cytomegalovirus / chicken beta-actin (ubiquitous strong promoter) HRdCVFL: Human RdCVFL cDNA BGH poly A: stabilized 3 'ITR AAV inverted terminal repeats of the mRNA. Density of cones (PN44) after subretinal injection of rd1 mice (PN14) of AAV2-GFP versus AAV2RdCVF / RdCVFL [CT35].

Example 1: In retinal degeneration, AAV-RdCVF rescues the cone and AAV-RdCVFL protects the rod.
Example 1 corresponds to experimental data published by the inventors of the following publications:

  Byrne LC, Dalkara D, Luna G, Fisher SK, Clerin E, Sahel JA, Leveillard T, Flannery JG., J Clin Invest., 2015 Jan; 125 (1): 105-16.doi: 10.1172 / JCI65654. Epub 2014 Nov 21. “Viral-mediated RdCVF and RdCVFL expression protects cones and rods in retinal degeneration” (32 in the list of references below).

  Through gene transfer into rd10 mice, a well-characterized model of rod cone dystrophy resulting from mutations in the β-subunit of PDE6 (rod-specific cyclic GMP phosphodiesterase). We describe experiments that study the bifunctionality of the NXNL1 gene by assessing the effect of expression of two RdCVF isoforms (RdCVF and RdCVFL) (14, 15). Retinal degeneration of rd10 is slower than that of rd1 mice, the most widely studied recessive retinal degeneration model that loses most of the photoreceptors by P15-P20. In rd10 mice, rod loss begins at P18 and peaks around P25, so the major stages of rod loss do not overlap with photoreceptor terminal differentiation (16). The rd10 mouse model is suitable for gene therapy (17, 18) and in this mouse model antioxidant treatment has been shown to delay rod loss (19). In addition, rearing in low light conditions has been shown to slow the rate of retinal degeneration, which opens up the potential for therapeutic treatment (17).

  Here, we examine the effect of AAV-mediated expression of RdCVF and RdCVFL. These studies use two routes of viral vector administration: systemic injection of AAV9 via the tail vein at P1, and intravitreal injection at P15. Early systemic injection is a transduction encoded by the AAV vector prior to the main stage of rod cell death and safely within the scope of assessing the effect of transgene expression on rod and cone degeneration. Allows gene expression to occur early in the degeneration process. Systemic delivery is not a clinically meaningful delivery mode to the retina. That is because many other tissues are infected simultaneously and the immune response is a major barrier to this approach. Therefore, we also have the effect of intravitreal injection (a commonly used technique for intraocular delivery) of a new variant of AAV called 7m8 that transduces photoreceptors from the vitreous. (20).

  We show here that the expression of two isoforms of RdCVF has a positive contrasting effect on rod and cone survival. Increased expression of RdCVF via systemic and intravitreal injections resulted in structural and functional rescue of cone photoreceptors, but had little effect on the rod. While RdCVFL itself did not significantly rescue the cones, co-expression of RdCVF and RdCVFL increased the observed rescue effect. In contrast, expression of RdCVFL early in the course of disease in rd10 animals housed in the dark maintained rod function, increased rhodopsin levels, and decreased cellular oxidative stress by-products.

  These results suggest that RdCVF and RdCVFL protect the photoreceptors through separate complementary mechanisms and suffer from various rod cone dystrophies regardless of the causative mutation. A proof-of-concept of widely applicable viral vector-mediated gene therapy that can sustain visual acuity in patients

Methods Animals C57B1 / 6J rd10 mice and P23H mice were obtained from Jackson Laboratories (Bar Harbor, ME) and were housed in a 12 hour light / dark cycle (except when moved to a dark box for dark rearing). All experiments were conducted using the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Office of Laboratory Animal Care at the University of California. (Berkeley, CA).

Production of viral vectors AAV vectors carrying cDNA encoding mouse-RdCVF, RdCVFL or eGFP were produced by the plasmid co-transfection method (31). Recombinant AAV was purified by iodixanol gradient ultracentrifugation and heparin column chromatography (GE Healthcare, Chalfont St Giles, UK). Virus eluent was buffer exchanged, concentrated in PBS with Amicon Ultra-15 Centrifugal Filter Units, and titrated against a standard curve by quantitative PCR.

Agarose section and confocal microscopy A new incision was made in the retina and immediately placed in 4% paraformaldehyde overnight at 4 ° C. Relief cuts were made and the entire retina was embedded in 5% agarose. A 150 μm transverse section was cut on a vibratome (VT 1000S, Leica Microsystems). The sections were then mounted on slides for confocal microscope (LSM710, Carl Zeiss; Thornwood, NY) with Vectashield mounting media (Vector Laboratories, Burlingame California).

Intravascular injection Children from day 1 after birth were fixed and the tail vein visualized using a surgical microscope. 10 μl of the vector solution was aspirated into a 3/10 cc insulin syringe. A 30 gauge needle was inserted into the vein and the plunger was manually depressed. A total of 5 × 10 ¹¹ DNase resistant particles were injected. It was verified that the injection was correct by focusing on the whitening of the veins. After injection, the children were allowed to recover for several minutes on a 37 ° C. thermal pad before returning them to the cage.

Intravitreal injection Mice were anesthetized with ketamine (72 mg / kg) and xylazine (64 mg / kg) by intraperitoneal injection. A 30 1/2 gauge disposable needle was passed through the equator of the anterior corneal limbus and the sclera behind it into the vitreous cavity. Then, while directly observing the needle just above the optic nerve head, a total volume of 5 × 10 ¹⁰ DNase resistant particles in a volume of 1 μl was injected into the vitreous cavity. The contralateral control eye received a vector carrying the gene encoding GFP or PBS.

Dark breeding A dark breeding mouse is born and raised in a light-safe box under low-intensity red light, and this box is opened for a short period of time for animal management, and this is opened with red light. Went under. Animals were placed in and out of the experimental box in a shielded cage.

qRT-PCR
Animals were humanely euthanized by CO ₂ overdose and cervical dislocation. One retina was collected from each mouse under each experimental condition (n = 5). RNA was extracted from each retina separately (RNeasy micro kit, Qiagen, Valencia, Calif.), Subjected to DNase digestion, and cDNA was prepared using the resulting RNA. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control and a non-RT control without reverse transcriptase was used to confirm the absence of genomic DNA. QRT-PCR was performed on the samples using verification primers for RdCVF or rhodopsin. MRNA levels were determined using the qRT-PCR relative standard curve method using the WT cDNA standard curve and expressed as a percentage of WT. Individual samples were run in triplicate as technical repeats.

Fundus imaging Fundus imaging was performed in anesthetized live mice with a fundus camera (Micron II; Phoenix Research Labs Inc., Pleasanton, Calif.) Equipped with a filter to monitor GFP expression. After application of proparacaine, the pupils were dilated with phenylephrine (2.5%) and atropine sulfate (1%) for fundus imaging.

ERG
Mice were dark adapted for 2 hours, then anesthetized and subsequently dilated of the pupil. Mice were placed on a 37 ° C. thermal pad and contact lenses were mounted on the binocular cornea. A reference electrode was inserted into the forehead and a ground electrode into the tail. For experiments on retinal function under dark field conditions, ERGs were recorded under flash intensity in the range of -3 to 1 log cd · s / m ² in the dark background (Espion E2 ERG system; Diagnosys LLC, Littleton, MA). Each stimulus was presented in a series of 3 flashes. For recording photopic ERG, mice were first exposed to a rod saturated background for 10 minutes. Stimulations ranging from -0.9 to 1.4 log cd · s / m ² were presented 20 times in a light background. Flicker ERG was obtained following presentation of a 30 Hz stimulus in a rod saturated background. Stimulus intensity and timing were computer controlled. Data were analyzed with MatLab (v7.7; Mathworks, Natick, MA). Student t test was used to compare ERG amplitude.

Mosaic acquisition and cone quantification Retina whole tissue specimens were obtained from normal donkey serum (Jackson ImmunoResearch Laboratories; PBS containing 0.5% BSA, 0.1% Triton X-100 and 0.1% azide (PBTA); West Grove, PA) (1:20) was blocked overnight at 4 ° C. and placed on a rotator for continuous stirring. Then goat anti-S-opsin (1: 100; Santa Cruz Biotechnologies; Santa Cruz, CA), rabbit anti-M / L-opsin (1: 500; Chemicon International; Temecula, CA), and mouse anti-rhodopsin (1: 100). An antibody cocktail containing (a gift from Dr. Robert Molday, University of British Columbia) was added to the solution of PBTA and incubated for 2 days. The retinal preparation was then washed 3 × 15 minutes and 1 hour in PBTA, after which the corresponding secondary fluorophore was added and incubated overnight at 4 ° C. Finally, the sample was rinsed, mounted, and cover slipped with Vectashield ((Vector Labs; Burlingame, Calif.). Olympus Fluoview 1000 scanning confocal laser microscope using a 40 × UPlanFLN (NA 1.3) oil immersion lens. (Center Valley, PA) were used to examine and collect images of the mouse retina using an automated stage (Applied Scientific Instrumentation: Eugene, OR) at 1 μm intervals on the z-axis and 1024 × in the xy direction. Optical sections were taken at a pixel resolution of 1024. These files were then used to create a maximum projection using the bioimage analysis software Imago (Santa Barbara, Calif.) 20% of the individual images Digital images were taken with the overlap of and the resulting approximately 300-400 images were synthesized using Imago, after which Imaris software (Bitplane AG, Zurich, Switzerland) and Pytho Cone counting was performed using custom software written in n.

TBARS Assay Malondialdehyde (MDA) concentration was determined using the TBARS assay (Cayman Chemical Company, Ann Arbor, MI, USA). Three retinas were pooled for each assay condition and the assay was repeated three times. A total of 25 mg of sonicated retinal tissue was used for each assay. The retina was sonicated in lysis buffer containing a protease inhibitor cocktail and then centrifuged. The supernatant was used for the TBARS assay and the assay was performed according to the manufacturer's instructions using technical replicates prepared in triplicate. A standard curve was constructed using known concentrations of MDA samples, and the concentration of MDA in the samples was determined against the resulting curves.

Statistics A two-tailed or unpaired Student t test was used for comparison of experimental groups. P values less than 0.05 are considered statistically significant and are indicated by an asterisk. P values less than 0.01 are indicated by double stars. The error bar displays the standard deviation.

Results Systemic Delivery of AAV92YF via Tail Vein Injection with P1 We have achieved early treatment by intravenous (tail vein) injection of a self-complementary AAV9 vector (AAV92YF) with two tyrosine to phenylalanine mutations. The therapeutic effect of RdCVF and RdCVFL delivery was investigated. While intraocular injection into the developing eye is not feasible at 1 week after birth, AAV92YF crosses the blood-retinal barrier when injected into the tail vein at P1, and high level gene expression throughout the retina (21). Systemic delivery leads to the development of expression in the retina much earlier in development, before a significant number of rods are lost. AAV92YF with a universal CAG promoter driving GFP expression (AAV92YF-scCAG-GFP) resulted in early retinal expression, which was obtained by in vivo fundus imaging in the retinal flat mount at P8 and immediately after opening at P15. It was visible. Fundus images with P35 showed strong GFP expression throughout the retina. Retinal flat mounts revealed that a large number of photoreceptors were transduced. At P35, GFP expression was observed in all retinal layers, ganglion cells, Müller glia, amacrine cells and photoreceptors, and RPE. Similar broad and strong expression patterns that were readily visible without immunolabeling were observed in the WT and rd10 retinas. QRT-PCR performed on mRNA collected from rd10 mice housed in a standard light-dark cycle in which P1 was injected with AAV92YF-scCAG-RdCVF, AAV92YF-scCAG-RdCVFL, or PBS It was revealed that the injection resulted in high levels of RdCVF expression in the retina at P35. As expected, RdCVF mRNA levels were greatly reduced in rd10 animals injected with PBS of the same age compared to WT (5% ± 5% WT). In contrast, the level of expression after AAV-mediated gene delivery was comparable to endogenous RdCVF levels in WT animals (AAV92YF-RdCVF = 79% ± 30% WT; AAV92YF-RdCVFL = 59% ± 13) % WT). Rhodopsin mRNA expression levels remained low throughout the condition, indicating that the rate of rod photoreceptor loss is similar in P35 treated and untreated animals (AAV92YF-RdCVF). = 8% ± 3% WT; AAV92YF-RdCVFL = 1% ± 1% WT; PBS 6% ± 1% WT).

  Transgene expression from AAV-mediated delivery is dose dependent. Animals injected with AAV92YF-scCAG-GFP 2E + 11, 5E + 11 or 1E + 12 virus particles and imaged one month after injection showed that GFP levels increased with increasing virus titer. In addition, qRT-PCR from animals injected with a broad range of AAV92YF-scCAG-RdCVF titers showed that RdCVF expression increased with more injections of viral particles.

AAV92YF. scCAG. Effect of injection of RdCVF Mice were intravenously injected with AAV92YF-scCAG-RdCVF at P1 and then housed in a standard light-dark cycle. The photopic electroretinogram (ERG) was then measured to determine the effect of RdCVF expression on cone function. A representative ERG trace illustrates the improved waveform and amplitude of ERG recorded from an eye injected with AAV92YF-scCAG-RdCVF compared to injection of AAV92YF-scCAG-RdCVFL, AAV92YF-scCAG-GFP or PBS. AAV92YF-scCAG-mediated expression of RdCVF was AAV92YF-scCAG-RdCVFL (46.7 ± 6.4 μV, p <0.005), AAV92YF-scCAG-GFP (46.6 ± 14.9 μV, p <0.01). ) Or PBS (56.5 ± 4.64 μV, p <0.01) resulted in significantly higher amplitude photopic ERGb waves (97.1 ± 10.67 μV). The WT b-wave amplitude was 156.6 ± 11.4 μV. ERG was recorded from 5 animals per condition. Data are presented as mean ± SD. The photopic flicker ERG was also recorded as a measure of cone function. A representative flicker ERG trace illustrates the improved amplitude and waveform compared to animals injected with GFP.

  The rescue of photopic ERG is dose dependent, indicating that the level of RdCVF expression required for significant rescue to occur should be achieved. The ERG amplitude was slightly increased with the injection of E + 11 vg, but the difference was not significantly different, whereas the injection of E + 12 vg resulted in a higher ERG amplitude.

  Transgene expression is persistent and GFP expression in animals injected with AAV9-scCAG-GFP can be easily visualized on a retinal flat mount imaged one year after injection. In rd10 animals injected with AAV92YF-scCAG-RdCVF, qRT-PCR reveals elevated levels of RdCVF one year after injection.

AAV92YF. scCAG. Cone density in animals injected with RdCVF Cone survival is quantified using automatic measurement of immunofluorescently labeled cone outer segments. The entire animal-derived retina previously used for ERG recording was flat mounted and stained with antibodies against S-opsin (blue label) and M / L-opsin (red label). High resolution 40 × z stack images were collected across the retina, registered, and grouped together to create a mosaic. Retina flat mount mosaics revealed a higher number of cones labeled with both S-opsin and M / L-opsin in all flat mounts from animals injected with AAV92YF-scCAG-RdCVF. A larger number of surviving cones of both types are seen in higher resolution images near the optic nerve head, the region of the retina with the most severe degeneration. Automatic quantification of cone density in retinas injected with AAV92YF-scCAG-RdCVF and PBS reveals significantly higher numbers per mm ² of both S-cones and M / L-cones in treated eyes I made it. S-cone density: RdCVF treated eye: 5573 ± 211 / mm ² ; PBS treated eye: 2,961 ± 917 / mm ² ; p <0.01, WT cone: 7446 ± 868 / mm ² . M / L-cone density: eyes treated with RdCVF: 8755 ± 1572 / mm ² ; eyes treated with PBS: 2682 ± 293 / mm ² ; p <0.01; WT cones 9761 ± 784 / mm ² Met.

Effect of systemic injection of AAV92YF-scCAG-RdCVFL Animals are housed in the dark to slow down the rate of rod loss and allow the development of RdCVFL expression in rods prior to apoptosis. Lowering the exposure slowed down the rate of photoreceptor denaturation as previously shown (17). At P1, mice were injected with AAV92YF-scCAG-RdCVFL, AAV92YF-scCAG-GFP or PBS (n = 6 for each group). Dark vision full field ERG was recorded weekly. Records from maximum intensity light stimulation performed at 3, 4 and 5 weeks after injection revealed a similar loss of a-wave amplitude at 3 and 4 weeks, but this improvement is no longer seen at 5 weeks. I couldn't. This difference was only statistically significant 4 weeks after injection (p <0.05). A more detailed analysis was performed on the second litter of mice injected with AAV92YF-scCAG-RdCVFL or PBS. In this group, the most significant difference in a-wave amplitude was observed at lower light intensity (-1 and -2 log cd · s / m ² ) 4 weeks after injection. A representative ERG trace illustrates the conservation of a and b wave amplitudes in animals injected with AAV92YF-scCAG-RdCVFL compared to control animals injected with GFP or PBS. ERG recording of photopic ERG revealed a delayed decrease in cone ERG in eyes expressing RdCVFL, which was most prominent after 5 weeks of injection, although this difference is not statistically significant There wasn't.

QRT-PCR for rhodopsin mRNA in dark reared animals injected systemically with AAV92YF-scCAG-RdCVFL
QRT-PCR performed on mRNA collected from P28 animals injected with AAV92YF-RdCVFL, GFP, or RdCVF at P1 and reared in complete darkness was at the rhodopsin mRNA level in animals injected with AAV92YF-RdCVFL. An increase was demonstrated (82% ± 21% WT, p <0.05), PBS (56% ± 6% WT), AAV92YF-GFP (59% ± 15% WT), or AAV92YF-RdCVF (57%) There was no increase at ± 16% WT). These results, along with rhodopsin mRNA levels measured in animals housed in a standard light-dark cycle, are expressed in a short period of time in this relatively fast retinal degeneration model (beginning of photoreceptor loss several weeks after birth) Shows the importance of RdCVFL delivery on the likelihood that the effects of. This is because no effect on the level of rhodopsin mRNA was observed in animals that were injected with AAV92YF-RdCVFL and reared in the light.

Measurement of lipid peroxidation Using the thiobarbituric acid reactive substance (TBARS) assay, lipid peroxidation in retinas treated with AAV92YF-scCAG-RdCVFL, AAV92YF-scCAG-RdCVF, AAV92YF-scCAG-GFP or PBS The level of product malondialdehyde (MDA) was determined. The test was performed on three pooled retinas and was repeated three times. MDA levels were reduced by 18% ± 0.9% in eyes treated with RdCVFL compared to untreated eyes.

Intravitreal injection of the novel viral variant 7m8 We characterized the viral affinity and expression pattern of the novel viral variant 7m8 in WT and rd10 mice after intravitreal injection on the 15th day after birth (P15). 7m8 is a variant of AAV2 developed to transduce the outer retina after intravitreal injection (20). Importantly, this variant transduced photoreceptors without subretinal injection, which was shown to cause injury responses and trophic factor release (22). Intravitreal injection of a self-complementary 7m8 vector encoding GFP at P15 resulted in strong expression one week after injection, which was also clearly visible in the fundus image at P45. Flat-mounted retinas showed that many photoreceptors were transduced by 7m8. Confocal imaging of retinal fragments revealed GFP expression in cells in the ganglion cell layer (GCL), inner granule layer (INL) and outer granule layer (ONL) in WT and rd10 mice.

  The level of RdCVF expression after 7m8 injection was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). MRNA levels in P45 rd10 mice injected with viral vectors encoding RdCVF, RdCVFL or GFP were quantified. As expected, RdCVF mRNA levels were reduced in control GFP injected rd10 mice compared to WT (4% ± 1% WT). Intraocular injection of 7m8-scCAG-RdCVF or 7m8-scCAG-RdCVFL with P14 resulted in higher levels of RdCVF mRNA. Levels were 127% ± 35% WT in animals injected with 7m8-scCAG-RdCVF and 91% ± 10% WT in animals injected with 7m8-scCAG-RdCVFL. Rhodopsin levels were uniformly low in all measured retinas (RdCVF = 3% ± 1% WT; RdCVFL = 1% ± 1% WT; PBS 1% ± 1% WT). Data are presented as mean + SD, n = 5 animals per condition.

Effect of RdCVF and RdCVFL expression on cone structure and function Next, we investigated the effect of 7m8 intraocular injection encoding RdCVF and RdCVFL on cone rescue in rd10 mice housed in the dark. . Injection of 7m8-scCAG-RdCVFL at P14 is not statistically significant with small photopic ERGb wave amplitude compared to eyes injected with PBS (65 ± 5 μV) or GFP (69 ± 7.3 μV) There was an increase (74 ± 4.8 μV). Injection of 7m8-scCAG-RdCVF significantly increased the amplitude of photopic ERGb wave amplitude compared to eyes injected with GFP or PBS (89 ± 7.9 μV, p <0.05). Simultaneous injection of 7m8-scCAG-RdCVF and 7m8-scCAG-RdCVFL resulted in significant rescue of photopic ERG, which was 53% higher than untreated eyes (99.75 ± 5.7 μV, p <0 .01), 17% higher than RdCVF alone (p <0.05). In all animals, one eye was injected with 7m8-scCAG-GFP or PBS as an internal control.

Cone labeling in eyes injected with 7m8-scCAG-RdCVF Anti-S and M / L-opsin antibodies were used to label cone populations in eyes injected with 7m8-scCAG-RdCVF or 7m8-scCAG-GFP. And a high resolution 40 × image mosaic was created. The labeling revealed that the number of cones increased most significantly in the central region of the retina near the optic nerve head. Automatic quantification of labeled cones across the retina compared to eyes injected with the opposite 7m8-scCAG-GFP (S-cone: 1254 ± 326, M / L-cone 1112 ± 419) Revealed an increase in cones expressing S- and M / L-opsin in eyes expressing 7m8-scCAG-RdCVF (S-cone: 1644 ± 436 p <0.05, M / L-cone : 2205 ± 264, p <0.05).

Effect of RdCVF and RdCVFL expression in P23H mice Next, 7m8 was used to deliver RdCVF or RdCVFL in homozygous or heterozygous P23H mice (dominant retinal pigment degeneration model). Quantification of ERG recording showed that injection of 7m8-scCAG-RdCVF resulted in an increase in photopic ERG amplitude compared to homozygous P23H / P23H mice injected with PBS (RdCVF: 39 ± 15 .7 μV, PBS: 19 ± 11.3 μV, n = 6, P <0.01). In heterozygous P23H / + mice, injection of 7m8-scCAG-RdCVF resulted in an increase in the amplitude of photopic ERG recordings up to 4 months after treatment compared to the contralateral eye treated with control GFP ( 1 month after injection, RdCVF: 175 ± 21.4 μV vs. PBS: 107 ± 17.2 μV, 4 months after injection, RdCVF: 71.5 ± 18 μV vs. PBS: 45 ± 15.6 μV, 6 months after injection, RdCVF: 23 8 ± 14.9 μV vs. PBS: 18 ± 10.4 μV). In contrast, treatment with RdCVFL did not result in any significant change in the amplitude of photopic ERG recording at any time point measured (1 month post injection, RdCVFL: 135 ± 47.3 μV vs. PBS: 120 25 ± 53.7 μV, 4 months after injection, RdCVFL: 72 ± 38 μV vs. PBS: 72.8 ± 45.2 μV, 6 months after injection, RdCVFL: 47.2 ± 55.8 μV vs. PBS: 31 ± 32 μV).

CONCLUSION We have demonstrated the effectiveness of AAV vectors as RdCVF delivery strategies and that RdCVF expression is a promising strategy to delay cone loss in patients with rod cone dystrophy Indicates.

  These results demonstrate for the first time functional photoreceptor rescue in the retina using AAV delivery via intravascular injection.

  Similar to systemic delivery of AAV92YF, 7m8 intraocular injection encoding RdCVF rescued cone function and sustained cone survival. Finally, the rescue effect observed after intravitreal administration of 7m8-scCAG-RdCVF was enhanced by co-administration of 7m8-scCAG-RdCVFL, a co-expressed gene therapy strategy that utilizes the synergistic activity of RdCVF and RdCVFL Suggests the potential.

  Taken together, these experiments support the role of NXNL1 as a bifunctional gene encoding two isoforms of RdCVF with different activities in the retina. Here, RdCVF has been shown to support cone survival, while RdCVFL has little direct effect on cones, but protects rod function through thiooxide reductase activity.

Example 2: Combination of RdCVF and RdCVFL produces synergistic effects The following constructs were produced and introduced into the AAV2 vector.

  Proviral plasmid p618 and its elements are described in the international patent application and publication (33) published as WO2012158757A1.

2xRdCVF: plasmid p857 and AAV CT-39
P857 / CT39 was designed to increase the level of RdCVF expression compared to CT-37 (RdCVF stuffer) to achieve sufficient cone protection in patients with retinitis pigmentosa (RP).

RdCVF-RdCVFL: plasmid p853 and AAV CT-35
This vector can co-express the short isoform and long isoform of RdCVF.

  Using these vectors, RdCVF and / or RdCVFL proteins were successfully expressed in porcine retinal pigment epithelium (RPE) cells. The protective effects of these cultures were evaluated in cultures rich in chick embryo derived cones and were very satisfactory.

  This construct was also tested for protective effect on the cones of rd1 mice after subretinal injection. A significant increase in cone density was observed (see FIG. 3).

  In conclusion, the inventors have shown that the combination of the short and long isoforms of the NXNL1 gene (RdCVF and RdCVFL) in a single expression vector has a synergistic effect in increasing in situ cone viability in the retina. Showed that to provide.

  The combinations according to the invention are useful for treating and / or preventing cone degeneration and other neuronal degeneration.

References Throughout this application, various references describe the state of the art in the field to which this invention pertains. The disclosures of these references are incorporated into this disclosure by reference.

Claims (14)

  A method for treating a patient suffering from a retinal degenerative disease, comprising: a first nucleic acid encoding a therapeutically effective amount of a short isoform of the NXNL1 gene, rod-derived cone survival factor (RdCVF) And administering a second nucleic acid encoding a long isoform (RdCVFL) of the NXNL1 gene.   The method of claim 1, wherein the first nucleic acid and the second nucleic acid are contained in a single expression vector.   An expression vector comprising a short isoform of the NXNL1 gene, a first nucleic acid encoding a rod derived cone survival factor (RdCVF) and a second nucleic acid encoding a long isoform of the NXNL1 gene (RdCVFL).   The expression vector according to claim 3, wherein the expression vector is a virus.   The expression vector according to claim 4, wherein the virus is an adeno-associated virus (AAV).   6. The expression vector according to claim 5, wherein the AAV is selected in the group consisting of AAV2, AAV9 and AAV7m8.   The expression vector according to any one of claims 2 to 6, wherein the short isoform of RdCVF is a short isoform of human RdCVF, and the long isoform is a long isoform of human RdCVFL.   The expression vector according to any one of claims 2 to 7, comprising a nucleic acid having a sequence as shown in SEQ ID NO: 1.   9. An expression vector according to any one of claims 2-8 for use in a method for treating a retinal degenerative disease.   The expression vector for use according to claim 9, wherein the retinal degenerative disease is retinal pigment degeneration.   11. Expression vector for use according to claim 9 or 10, which is for administration by intravenous injection or by intravitreal injection.   The method of claim 1, wherein the first nucleic acid and the second nucleic acid are contained in separate expression vectors. A kit of parts for use in a method for treating a photoreceptor degeneration disorder, comprising:
A first expression vector comprising a first nucleic acid encoding a rod-derived cone survival factor (RdCVF), and a second encoding a long isoform of the NXNL1 gene (RdCVFL) A kit of parts comprising a second expression vector comprising a nucleic acid.   The method according to claim 12 or the kit of parts according to claim 13, wherein the expression vectors are administered simultaneously or sequentially.